This is an R01 renewal application to study the mechanisms and genetics of immune tolerance in mice expressing B cell receptor-reactive superantigens. In the previous funding period, we generated and studied a large series of transgenic mice carrying custom designed superantigens reactive to immunoglobulin constant regions. Single chain antibodies reactive to Ig C regions were generated by engineering monoclonal antibody genes from hybridomas. These combining sites were expressed as chimeric membrane proteins, called macroself antigens (Ags), in transgenic mice. Macroself Ag- expressing mice were then analyzed for perturbations of lymphocyte survival, function, and receptor gene usage. Macroself Ag transgenics with specificity for IgM, Ig-kappa, IgD, and IgG2a allowed analysis of immune tolerance in different cell compartments and developmental stages in a non-clonal immune system. The utility of using macroself Ag mice as recipients of bone marrow to rapidly study the phenotypes of mouse mutants was also demonstrated. In this grant we propose to follow up these findings by focusing on two novel strengths of the experimental system-- the ability to conveniently study tolerance in B cells that have undergone an IgG class switch and the ability to impose a stringent in vivo screen for cells manifesting defective immune tolerance. In the first Aim we propose to characterize how IgG2a B cells are rendered tolerant in IgG2a-macroself Tg mice and to generate and analyze tolerance in IgG1-macroself Tgs. Candidate mouse mutants will be screened for defects in immune tolerance in the IgG compartment. In the second Aim, we will take advantage macroself Tg mice to impose selections in order to reveal tolerance defects in B cells with candidate or random mutations. We will take advantage of a random insertional mutagenesis screen to identify genes regulating tolerance. The long term goal of these studies is to understand the genetic basis of immune tolerance and autoimmunity.